Optical waveguides have been used in various analytical test. For example, in an article entitled "Optical Fiber Fluoroprobes in Clinical Analysis", Clin. Chem, 29/9, pp 1968-1682 (1983), Michael J. Sepaniak et al. describe the use of quartz optical fluoroprobes. By incorporating a single fiber within a hypodermic needle, the authors have been able to obtain in vivo measurement of the fluorescence of various therapeutic drug analytes in interstitial body fluids. Sepaniak et al state that their probe must use a laser radiation source as a fluorescence exciter.
One of the fluoroprobe designs uses a capillary action design for sampling. A length of optical fiber is stripped of its protective coating and slid inside a standard glass capillary tube, touching the walls of the capillary tube at random but not extending the whole length of the tube. This assembly is placed within a hypodermic needle.
Immunoassays using optical waveguides have been disclosed in European Patent Applications 82201107.8 and 81810385.5 to Battelle Memorial Institute. The earlier 1981 application discloses a competitive immunoassay using fiber optics. More particularly, a glass single or multimode optical fiber having a core with an index of refraction (N.sub.1) and a cladding with an index of refraction (N.sub.2), where N.sub.1 &gt;N.sub.2, is coated with an antibody (A.sub.b) film to form a sensor fiber.
The immunoassay is done in three steps. First the sensor fiber is immersed into a fluid containing an antigen (A.sub.g) analyte specific to A.sub.b plus a known amount of fluorescent-labelled A.sub.g. A fluorescent coating forms in proportion to the A.sub.g concentration. Then, an excitation radiation is propagated down the sensor fiber core at one end. The immunoassay relies upon "evanescent wave" phenomena, i.e., the electromagnetic field components which extend a short distance into the cladding, to interact with and excite the external A.sub.b /tagged A.sub.g complex. Finally, fluorescence from the excited tagged complex is "reinjected" back into the propagated down the core where it is detected at the opposite end of the fiber. The fluorescence may be reflected and emerge from the output end where it can be separated and detected.
In a continuation-in-part application filed in 1982, Battelle describes how to control the penetration of the exciting evanescent wave into the analyte-containing fluid. Here, the index of refraction of the core N.sub.1 is greater than that (N.sub.2) of the fluid such that the ratio N.sub.1 /N.sub.2 permits the evanescent wave to penetrate only to the thickness of the A.sub.b /A.sub.g complex. Thinner layers of such a complex are said to require an index of refraction which would eliminate a glass cladding. The second Battelle application includes more types of immunoassay examples using fiber optics, specifically, "sandwich," "limited reagent," "direct," and "sequential saturation" immunoassays.
An immunoassay apparatus developed by T. Hirschfeld is disclosed in U.S. Pat. No. 4,447,546 issued May 8, 1984, which employs total internal reflection at an interface between a solid phase and a fluid phase of lower index of refraction to produce an evanescent wave in the fluid phase. Fluorescence excited by the wave is observed at angles greater than the critical angle, by total reflection within the solid medium. The solid phase is arranged and illuminated to provide multiple total internal reflections at the interface. Typically, the solid phase is in the form of an optical fiber to which is immobilized a component of a complex formed in an immunochemical reaction. A fluorophore is attached to another component of the complex. The fluorescent labeled component may be either the complement to or the analog of the immobilized component, depending upon whether competive or sandwich assay are to be performed. In the case of competitive assays, the labelled component is typically preloaded to the immobilized component in a controlled concentration.
The fiber and the attached constituent of the assay are immersed in a fluid phase sample and the exciting illumination is injected into an input end of the fiber. The evanescent wave is used to excite fluorescence in the fluid phase, and that fluorescence which tunnels back into the solid phase (propagating in direction greater than the critical angle) is detected at the input end of the fiber.
The observed volume of sample is restricted not only by the rapid decay of the evanescent wave as a function of distance from the interface, but by an equally fast decrease with distance of the efficiency of tunneling, the more distant fluorophores not only being less intensely excited and thus fluorescing less, but their radiation is less efficiently coupled into the fiber. Consequently the effective depth of the sensed layer is much reduced compared to the zone observed by total reflection fluorescence alone, the coupling efficiency effectively scaling down the zone.
Multiple total internal reflections in the solid phase allow the illuminating beam to excite repeatedly an evanescent wave, thereby more efficiently coupling the small excitation source to the sample volume. This also increases the amount of sample sensed. The latter is also enhanced by diffusive circulation of the sample past the fiber surface and to which the material being assayed adheres by reaction as it passes. Diffusion makes the actually sampled layer thickness much larger than the thin surface layer.
All of the radiation that tunnels back into the fibers is within the total reflection angle, and is thus trapped within the fiber. The power available from the fluorescence increases with the length of fiber within the fluorescing material. However, the optical throughput of the system (determined by the aperture and the numerical aperture of the fiber) remains constant. The total fluorescent signal coming from the entire surface of the fiber, multiplied by the increase in sample volume due to diffusion, thus becomes available in a very bright spot (that is the cross-section of the fiber in diameter) exiting the fiber at its input end through a restricted angle determined by the critical angle of reflection within the fiber. Such signal is easily collected at high efficiency and throughput when matched to a small detector.